Depth selective ph measurement and uv exposure measurement

ABSTRACT

A Raman spectrum is measured inside animal tissue, such us human skin tissue, at a selected depth from a surface the tissue. A pH value is computed using a function that assigns a pH value as a function of the measured Raman spectrum. The computation may involve computing a number representing a ratio of concentrations of a protonated and a deprotonated version of a chemical substance from the Raman spectrum and generating pH information on the basis of said number. The chemical substance is for example a form of Urocanic acid (UCA). UV exposure is measured from the weight of the spectrum of cis-UCA.

FIELD OF THE INVENTION

The invention relates a method and apparatus for depth selective pHmeasurement in animal tissue (animal tissue being understood to includehuman tissue). The invention also relates to a method and apparatus formeasuring effects of exposing skin tissue to UV irradiation.

BACKGROUND ART

-   -   pH, or more generally the concentration of free Hydrogen atoms,        is an important parameter of tissue such as skin. pH at the skin        surface differs from individual to individual and is moreover        dependent on external influences such as the application of        cleansing products or other personal care products, such as        deodorants. Cleansing products (water, soaps, shampoos etc) can        have a pronounced effect on skin pH which only slowly returns to        the pH-value before cleaning. In addition there is a large        difference between pH of the surface of skin and the pH of        deeper layers of the skin, because a large difference in pH        exists between the skin surface and the vital epidermis. This pH        difference is maintained by the stratum corneum which is the        outermost skin layer. For the development of such products it        would be desirable to monitor their effects on the pH of the        skin.

Known methods of measuring pH involve for example the application ofchemical pH indicators or electrical measurements, wherein the potentialdifference between an electrode on the skin and a reference electrode ismeasured. An article by K. I. Mullen, D. Wang, L. G. Crane, and K. T.Carron, titled “Determination of pH: SERS Fiber Optic probes”, publishedin Analytical Chemistry 64, page 930 (1992) describes a technique formeasuring pH in water using a pH indicator molecule attached to the endof an optical fiber. Raman scattered light from the molecule is gatheredthrough the fiber and analysed. With these presently available methodsnon-invasive skin pH measurements with the known methods are limited tothe skin surface. Such measurements are unsatisfactory because of thelarge difference between pH of the surface of skin and the pH of deeperlayers of the skin. Invasive techniques are needed to measure the pHbelow the surface of the skin with these known methods, or moregenerally to measure pH as a function of depth in the skin. Thedisadvantage of invasive techniques is that the pH may be affected bythe invasive technique and/or that otherwise skin physiology isdisturbed. Moreover it is difficult to monitor ongoing changes in pH.

Accordingly, there exists a need for a method of measuring pH atselected depths in skin tissue without using invasive techniques, sothat the normal chemical processes in the skin are not influenced by themeasurements.

An article titled “In Vivo Confocal Raman microspectroscopy of the Skin:Non-invasive Determination of Molecular Concentration Profiles”published in March 2001 in the Journal of Investigative Dermatology 116pages 434-442 (2001), and authored by Peter J. Caspers, Gerald W.Lucassen, Elizabeth Carter, Hajo Bruining and Gerwin J. Puppelsdescribes depth selective Raman spectroscopy of skin tissue. Thisarticle is incorporated herein by way of reference.

Raman spectroscopy is a non-invasive technique that involvesilluminating material with essentially monochromatic light of a firstwavelength and observing the intensity of light that has beeninelastically scattered by the material, as a function of wavelength ofthe inelastically scattered light. The spectrum of the inelasticallyscattered light is a composite of contributions of the differentchemical species in the material.

In principle, each chemical species provides its own characteristiccontribution to the spectrum, in proportion to its concentration. Thismakes it possible to determine at least the relative concentrations ofchemical species in the material. Raman Spectroscopy can be made depthand/or location sensitive by focussing the monochromatic light at acertain depth or location and/or gathering inelastically scattered lightselectively from a depth or location.

Unfortunately, free protons do not have a measurable Raman spectrum.Therefore Raman spectroscopy cannot be used to measure light that isinelastically scattered by protons. However, the article mentions adiscovery by the inventors of the present invention that among a greatmany other contributions the Raman spectrum of light scattered by skintissue naturally contains a detectable contribution of UCA (Urocanicacid). The concentration of UCA in the stratum corneum depends on manyvariable factors. In addition the protonation of UCA variessignificantly in the pH range that may be encountered in the skin (pH'stypically range from 4.5 to 7). The Raman scattering spectrum of UCA ispH dependent.

SUMMARY OF THE INVENTION

Amongst others it is an object of the invention to provide for a methodand apparatus for depth selective measurement of pH in animal tissue andin particular in human skin tissue which leaves the tissue intact.

Amongst others it is an object of the invention to provide for a methodand apparatus for depth selective measurement of pH in animal skintissue.

Amongst others it is a further object of the invention to provide for amethod and apparatus for depth selectively measuring pH in animal tissueas a function of position transverse to the depth in the tissue.

Amongst others it is an object of the invention to make use of thepublished discovery that the Raman spectrum of light scattered by skintissue contains a contribution of UCA that is strongly pH dependent.

Amongst others it is another object of the invention to provide for atechnique for measuring an indication of a dose of UV irradiationreceived by skin tissue.

Amongst others it is another object of the invention to provide for atechnique for measuring an indication of potential health damage by UVirradiation received by skin tissue.

Amongst others it is another object of the invention to provide for atechnique to determine the efficacy of sun protection devices or sunscreens.

According to one aspect of the invention a Raman spectrum is obtainedfrom in a depth dependent way from inside the skin, for example at aselected depth by focussing a lightsource at the selected depth, or froma selected range of depths. From the measured spectrum a pH value iscomputed using a function that assigns a pH value as a function of themeasured Raman spectrum.

pH information is computed on the basis of said number. The function maycompute for example a ratio of the weights with which pH dependentspectra of a chemical constituent of the skin at different pH valuescontribute to the measured spectrum and a pH value derived from thisratio is output. Although Raman spectroscopy requires cumbersomecalibration to determine absolute concentrations from the weights, thedetermination of ratio's does not require that calibration. The computedpH value may be output for display to a human operator, in the form of anumber or as a graph of pH versus depth, so as to enable the operator toevaluate skin conditions for example after application of therapeutic orcosmetic substances to the skin or to diagnose skin diseases. Thecomputed pH value may also be used for example for automatic dosage ofapplication of substances to the skin, to switch supply of one or moreof the substances on or off dependent on the measured pH at a certaindepth in the skin, or to control the concentration with which thesubstances are supplied dependent on the measured pH.

Preferably, the concentrations of the protonated and deprotonatedversion of a form of urocanic acid (UCA), more preferably trans-urocanicacid, are used to compute the pH value from the spectrum but in otherembodiments cis-urocanic acid or histidine or any other tissueconstituent that has a pH dependent spectrum in the range of pH valuesthat occur in the tissue may be used, or a constituent comprising amixture of such substances may be used.

In a preferred embodiment a ratio of concentrations of protonated anddeprotonated versions is computed by fitting a set of weights with whicha number of predetermined spectra of contribute to the measuredspectrum, the predetermined spectra at least including two spectra of aconstituent that has a pH dependent spectrum, the two spectracorresponding to different pH values. Preferably, the predeterminedspectra also include Raman spectra of major chemical components thathave been found to occur in the type of tissue under study.

However, it is not necessary to use explicit spectra nor is it necessaryto use explicit fitting. It has been discovered that pH can be computedusing a function of the set of measured intensities that represent theRaman spectrum of animal tissue. Fitting is merely one way of definingthis function. As an alternative this function may be computed withoutfitting. For this purpose one may use an approximate function of theintensity values, whose input/output relation may be tuned by adjustinga number of adjustable parameters. The parameters are set in advance sothat the result of the function approximates the pH value given ameasured spectrum. Such a function will generally compute the pH fromthe entire spectrum, not just from the contribution of a singlechemical, like UCA.

Similar techniques may be used to measure health damaging effects of UVirradiation. According to another aspect of the invention a method ofmeasuring a health damaging effect of UV irradiation in skin tissue, isprovided that comprises

-   -   measuring a Raman spectrum of a part the tissue selected        dependent on a depth from a surface of the tissue;    -   computing an effect of UV irradiation using a function of UV        irradiation dependent aspects of the measured Raman spectrum.

It has been found that the Raman spectrum of skin tissue depends in ameasurable way on the relative concentrations of cis-UCA and trans-UCA.Cis-UCA is formed from trans-UCA under the influence of UV irradiation.The concentration of cis-UCA is suspected to be arm agent in healthdamaging effects of UV irradiation. At least it is strongly correlatedwith these health damaging effects. Hence Raman spectroscopy providesfor a method and apparatus that can help people to avoid health damagedue to UV irradiation by providing a monitor for the rise of suchdamage. All embodiments of the method and apparatus for measuring pHapply mutatis mutandis to measurement of health damaging effects of UVirradiation as well. Also similar computer programs may be used mutatismutandis for computing the health damaging effect.

These and other objects and advantageous aspects of the method andapparatus according to the invention will be described in more detail ina non-limitative way using the following drawing

FIG. 1 shows a depth selective pH measuring apparatus

FIG. 2 shows a Raman spectrum of human skin tissue

FIG. 3 shows a structural formula of trans-UCA

FIG. 4 shows Raman spectra of UCA

FIG. 5 shows a flow-chart of a method of measuring pH

FIG. 6 a,b show resulting measurements as a function of depth

FIG. 7 shows structural formulas of cis-UCA and trans UCA

FIG. 8 shows Raman spectra of cis-UCA and trans-UCA

FIG. 1 schematically shows an apparatus for selectively measuring pH inskin tissue 13. The apparatus comprises a light source 10, a heightadjustment unit 11, a focussing lens 12, a splitter 14, an output filter15, a spectrometer 16, a computing device 17 and an output device 18.The light source 10 is arranged to illuminate the skin tissue 13 viasplitter 14 and lens 12. Height adjustment wait 11 is arranged to adjustthe position of lens 12 relative to skin tissue 18. Spectrometer 16 isarranged to receive scattered light from skin tissue 13 via lens 12,splitter 14 and output filter 15. Spectrometer 16 has an output coupledto computing device 17, which in turn has an output coupled to outputdevice 18.

The arrangement of a light source, focussing lens, splitter 14, outputfilter 15 and spectrometer forms a conventional Raman spectrometerarrangement. Any variation of such an arrangement may be used. Forexample various additional elements such as mirrors and filters may beadded to in the light path in light source 10, spectrometer 16 orbetween these devices. For example the light path may contain imagingoptics (not shown) to form an image of the skin at the depth at whichthe laser light is focused in an image plane. An aperture (not shown)may be provided in the image plane, such that the location where lightfrom light source 10 is focused in the skin 18 is imaged onto theaperture. The size and/or shape of the aperture may be selected to limitthe measurement volume to a well defined location in the skin. Lightpassed by the aperture is fed to spectrometer 16. Preferably amulti-channel detector is used in spectrometer 16, to measure theintensity of scattered light for a plurality of wavelength channels inparallel.

If desired one or more optical fibers may be used in part or all of thelight path from source 10 to skin 13 and from there back to spectrometer16, to conduct the incoming light and/or the scattered light.

Computing device 17 may be any suitably programmed computer. The programwith instructions to cause the computer to process the Raman measurementmay be a firmware program present in non-volatile memory in thecomputing device 17, or a program loaded from a program carrier such asa CD-ROM, a floppy disk etc.

In operation, essentially monochromatic light from light source 10 isfocussed onto skin tissue 13 by lens 12. The light source is preferablya monomode laser with an emission wavelength between 680 nm and 860 nm.Height adjustment unit 11 is adjusted so as to focus the light at aselected depth. Light scattered by skin tissue 13 from a location wherethe light is in focus is id to spectrometer 16. Elastically scatteredlight, that is, light with the same wavelength as the main wavelength oflight source 10 is filtered out by output filter 15. Spectrometer 16analyses the spectrum of the inelastically scattered scattered light,producing a collection of light intensity measurements, each for arespective wavelength. Computing device 17 reads the results of thelight intensity measurements from spectrometer 16.

FIG. 2 shows a typical example of a measured spectrum of inelasticallyscattered light as a function of wavelength (expressed in terms ofwavenumber shift, which defined as (1/λsource−1λ)*10⁷) where λ [in m]isthe wavelength of the inelastically scattered light) measured with lightfocused at a depth 70 micrometer below the surface of the skin 13. Itwill be observed that the spectrum contains a considerable amount ofstructure. This structure is characteristic of the mixture of chemicalsubstances present in the region where light is inelastically scattered.

It has been discovered that in skin tissue one of these substances thatcontribute to the structure of the spectrum is Urocanic acid (UCA). UCAis formed in the skin from Histidine under influence of the enzymeHistidase and diffuses from the vital epidermis into the stratumcorneum. Therefore the concentration of UCA in the stratum corneumdepends on many variable factors. UCA is also present in the liver andmay be used to measure pH inside liver tissue.

FIG. 8 shows a structural formula of the protonated form oftrans-Urocanic Acid (UCA). The acidic properties of UCA are mainly dueto a proton (H) bound to an imidazole ring. In the pH range of interestin the skin (4 to 7.5) trans-UCA has a pK value of 6.1 for protonationof the imidazole ring. At the lowest pH values that may occur in skintissue most of UCA remainder molecules have a proton bound to theirimidazole ring. At the highest pH values that occur in skin tissue theimidazole rings of most UCA remainder molecules are deprotonated.

FIG. 4 shows Raman inelastic scattering spectra of isolated trans-UCAmeasured at pH values of 4.5 and 7.5. It can be seen that the spectradiffer. Comparing FIG. 4 with FIG. 2 will show that trans-UCA is not thesole contributor to the Raman spectrum of skin tissue (it must be notedthat the peak between 1600 and 1700 cm-1 in FIG. 2 is only partly due tocontributions of UCA: it does not change as pronouncedly with pH as thepeak in the spectrum of UCA). Also the pH dependence of the Ramanspectrum due to the pH dependence of UCA is quite small. Nevertheless ithas been realized that the spectrum of UCA can be used to measure pH inskin tissue.

Computing device 17 is programmed to compute the pH value of the skintissue at the depth where the light is inelastically scattered from themeasured Raman spectrum of the inelastically scattered light.

FIG. 5 shows a flow-chart of a method of measuring pH in skin tissue. Ina first step 51, computing device reads information about the spectrumfrom spectrometer 16. Computing device 17 pre-processes the informationto correct for known wavelength dependency of the sensitivity of theinstrument (involving for example throughput through the light path andthe efficiency of spectrometer 16), and associates measured spectralintensity values with respective wavelengths.

In a second step 52, computing device 17 determines the weights withwhich different Raman scattering spectra of different chemical speciesthat are expected to be present in the skin tissue contribute to themeasured spectrum. For this purpose, computing device 17 stores datadescribing spectra of a number of such chemical species, including thespectrum of trans UCA at different pH values, for example pH values of4.5 and 7.5. Known spectra may be used to provide the stored spectra forthis purpose or the stored spectra may be provided by measuring spectraof UCA at different pH during a calibration stage. Instead of spectrameasured at different pH values, computed spectra may be used that formextrapolations of spectra measured at different pH values. Otherchemical species for which computing device 17 stores spectra, becausethey have been found to occur at one time or another in relevantquantities in the skin include pyrrolidone carboxylic acid, arginine,ornithine, citrulline, serine, proline, glycine, histidine, alaninine,lactate, urea, water, keratine, ceramides and cholesterol. Preferablyspectra of all these compounds, or combinations thereof (when thecompounds occur in predetermined ratio's) may be used during fitting.

The computing device 17 preferably assigns weights to each of the storedspectra by means of a least square fit, selecting a set of weights thatminimizes a squared measure of difference between the measured spectrumand a weighted sum of the stored spectra. Thus, in the second step 52the computing device 17 generates, amongst others, weights associatedwith spectra of UCA taken at different pH values.

In third step 53 computing device 17 computes a ratio of theconcentrations of the protonated and deprotonated form of UCA from theweights computed in second step 52. For example, when stored spectra offor the same concentration of UCA at pH values of 4.5 and 7.5 are usedto fit the measured spectrum (at which values UCA is nearly completelyprotonated and deprotonated respectively), the ratio R may be computedfromR=(w1*C1)/(w2*C2)

-   -   where w1 and w2 are the weights computed for the two stored        spectra of UCA and C1 and C2 are the concentrations of UCA in        the samples from which the stored spectra where obtained. Of        course, instead of spectra obtained at pH values where UCA is        nearly completely protonated and deprotonated respectively,        spectra at other pH values may be used. If a first spectrum is        assigned a weight wa and corresponds to concentrations Ca1 and        Ca2 of the protonated and deprotonated version respectively, and        a second spectrum is assigned a weight wb and corresponds to        concentrations Cb1 and Cb2 of the protonated and deprotonated        version respectively, then the ratio R may be computed from        R=(wa*Ca1+wb*Cb1)/(wa*Ca2+wb*Cb2)

Similarly, if the reference spectra that are weighed to fit the measuredspectrum correspond to mutually different but known concentrations, theweights are corrected proportionally to the known concentrations of thereference spectra. In fourth step 54 computing device 17 computes the pHvalue from the ration R and the known pK value (6.1) of UCA:

-   -   pH=pK+log(R).

Computing device 17 outputs the measured pH value to output device 18for further use. Output device 18 may output the pH value on a displayscreen that displays the measured pH value, but any other use may bemade of the measured pH value. For example, the measured pH value mightbe used to control dosage of material applied to the skin surface.

Although the invention has been described by way of example using thealgorithm of FIG. 5, it will be appreciated that many variations arepossible that all allow computation of pH from the measured spectrum.For example, one might directly fit R or even the pH, instead of usingthe weights w as intermediates. In another example, instead of fittingthe entire spectrum only part of the spectrum may be fitted or differentparts of the spectrum may be weighted differently for example accordingto the extent to which they depend on pH.

Although the invention has been explained in terms of measurements usingthe pH dependence of the Raman spectrum of trans-UCA, other substancesmay be used. Trans UCA is preferred because it has been found to beavailable in sufficient concentration in skin tissue. However, withoutdeviating from the invention pH dependent spectra of other materials maybe used, such as for example the protonated and deprotonated form ofhistidine, or of cis-UCA. These materials have also been found suitablefor measuring pH in skin tissue under some circumstances. One may evenuse a combination of these substances to determine pH.

The method described in FIG. 5 defines a function that assigns a pHvalue as a function of the intensity values of the measured spectrum. Ofcourse, the procedure of fitting weight values in a weighted sum of anumber of predetermined spectra to minimize the difference with themeasured spectrum, followed by computing pH from the weight values oftwo specific spectra is merely one way of computing this function. Inyet another example this function is computed without explicit fit. Forthis purpose an approximate function may be used with a number ofadjustable parameters. The parameters are set in advance so that thefunction approximates the ratio R given a measured spectrum.

Many ways of defining such an approximate function exist. For example,one may use a function defined by the operation of a computerized neuralnetwork. In this case a conventional neural network “training” proceduremay be used to select the parameters of the function so that the networkcomputes the pH value. In this case, a number of spectra taken atdifferent pH values is used in the training procedure, together with thecorresponding pH values, which may be obtained for example using theminimization method described in the preceding, or by other types ofmeasurement, or from information about the preparation of the specimenfrom which the spectra where taken.

But of course, instead of the functions that simulate a neural network,any other type of parameterised function with appropriately selectedparameters may be used, such as for example a polynomial or a ratio ofmultinomials (a multinomial is a function whose dependence on each ofthe various intensity values from the measured spectrum is polynomial)etc. Whatever function is used to assign pH, it will be appreciated thatthus the function will generally compute the pH from the entirespectrum, not just from the contribution of a single chemical, like UCA.

Preferably computing device 17 is coupled to height adjustment unit 11to control the depth of the skin at which light from light source 10 isfocussed. In this case, a scan of pH versus depth may be made, byrepeatedly adjusting the position of lens 12 relative to skin 13,measuring an inelastic scattering spectrum and computing pH according tothe steps of FIG. 5.

FIG. 6 a shows examples of weight values (in arbitrary units) computedfor the two UCA spectra as a function of depth D (distance to skinsurface) and FIG. 6 b shows the corresponding pH values.

Preferably, the apparatus of FIG. 1 is also provided with a mechanismfor scanning the measurement position transverse to the directionperpendicular to the skin. This may be realized using scannable mirrors.Such a transverse position and depth dependent measurement may becombined with other type of transverse position dependent measurement,such as for example simple optical imaging, so as to make it possible tocorrelate pH variations with other observable phenomena.

FIG. 7 shows structural formulas of cis-UCA and trans-UCA (cis-UCA is anisomer of trans-UCA). Trans-UCA is affected by UV (Ultra Violet)irradiation: under the influence of UV light trans-UCA is converted intocis-UCA. Conversion of trans-UCA into cis-UCA in the stratum corneum ofthe skin is thought to be an important agent in the health damagingeffect of UVB irradiation inside the skin. A higher ratio (relativelymore cis-UCA) occurs upon exposure to UVB irradiation. After irradiationthe ratio drops only slowly: it is assumed that the health damagingeffects persist until the ratio has dropped sufficiently. In any case,increases in the ratio of the cis-UCA concentration and the trans-UCAconcentration inside the skin is strongly correlated with healthdamaging effects of UV light.

It has been found that cis-UCA formation can be determined from theRaman spectrum of the skin.

FIG. 8 shows Raman spectra of trans-UCA and cis-UCA. The overall Ramanspectrum of skin tissue depends detectably on the ratio of theconcentration of cis-UCA to the concentration of other substances in theskin. Therefore, measurement of this ratio using depth selective Ramanspectroscopy can advantageously be used to monitor health damagingeffects of UV irradiation in a non-invasive way.

An apparatus and process for performing this measurement works basicallyin the same way as the apparatus and process for pH measurement, butinstead of the weights assigned to the spectra of the protonated anddeprotonated version of trans-UCA the weights assigned to the spectra ofcis-UCA and a reference substance (preferably trans-UCA) are used todetermine information about the concentration of cis-UCA. Of course,instead of fitting an adapted function may be used for this purpose asdescribed for the pH measurement.

The information about the concentration of cis-UCA is used to asses theextent to which UV irradiation has affected the skin tissue.

Any reference substance may be used but preferably the combination ofthe spectra of the protonated versions of cis-UCA and trans-UCA or thedeprotonated versions of cis-UCA and trans-UCA are used for this. Thisallows for the elimination of pH dependent errors since the logarithmsof the ratio of the concentrations of overall cis-UCA and trans-UCAdiffers from the logarithm of the concentrations of (de-)protonatedcis-UCA and trans-UCA by the predetermined difference of the pK valuesof cis-UCA and trans-UCA (pKcis-pKtrans=0.9). Moreover, since cis-UCA isformed from trans-UCA under influence of UV light, the use of trans-UCAas a reference eliminates the effect of individual differences in transUCA concentration on the measurement of the effect of UV irradiation.

If desired, the apparatus may select between using a measured ratio ofthe protonated concentrations or of the de-protonated concentrations,dependent on pH or rather on which version (protonated or deprotonated)yields larger weight values.

Such an apparatus or process that measures information about theconcentration of a chemical substance that is generated under theinfluence of UV irradiation may be used to perform various furtherfunctions. For example, in a device for use by people who takesun-baths, the measured concentration ratio may be used to trigger andalarm signal such as an audible signal or a warning light when the ratiopasses a predetermined threshold. The user is thereby enabled toterminate the sun-bath when there is more than a predetermined risk ofhealth damage, or to apply a sun tan lotion with a strong blockingfactor. Such an apparatus with an alarm may be part of an electricsolarium, or it may be a stand alone device that may also be usedoutdoors, for example on the beach. More generally such an apparatus maybe used by anyone that is exposed to UV irradiation, such as people thatare outdoors for their work, to determine whether action should be takenagainst health hazards (e.g. by using sun blocking substances or ceasingto work outdoors until the ratio drops). The threshold for generatingthe alarm signal may be set to any value that has been determined to beindicative of potential health damage, either for the individual usingthe device or in general. The exact value of the threshold depends onthe acceptable risk and may be set for example in future governmentalhealth standards.

Similarly, the apparatus might have an output for outputting a gradedsignal derived from the measurement of the ratio of concentrations oftrans-UCA and cis-UCA, for example in the form of a number of digitsthat represent a number indicative of the ratio, but other forms ofsignalling could be used, such as a meter with a pointing hand that ismade to deviate according to the measured ratio, or even a displayscreen in which the color of an area is controlled dependent on theratio, varying e.g. from green to red as the amount of cis-UCAincreases. The output of the graded signal can be used for example toselect a required blocking factor of sun-tan lotion, or to plan futureexposure to sunlight.

Of course this process and apparatus for measuring an effect of UVirradiation can also be used in experiments to determine the effectivityof sun tan lotions, or more generally of substances that are used toprotect the skin against the effects of UV irradiation. In this case oneapplies the substance to a skin area, irradiates the skin area with UVlight and subsequently measures the ratio of the cis-UCA and trans-UCAconcentration using Raman spectroscopy as described above. Preferablyanother area of the skin (preferably adjacent the first mentioned area)is treated similarly, except that the substance is not applied to theother area. The ratio's of cis-UCA and trans-UCA concentration measuredfor the two area's can then be compared to establish the differentialeffect of the substance. Of course a similar technique may be used in anapparatus for sun-bathers, by covering a part of the skin and measuringthe concentration in the covered part and an uncovered part of the skin,to generate an alarm signal or a graded indication of UV effects.

1. A depth selective method of measuring pH inside animal tissue, themethod comprising measuring a Raman spectrum of a part of the tissue,said part being selected dependent on a depth from a surface of thetissue; computing a pH value using a function that assigns a pH value asa function of pH dependent aspects of the measured Raman spectrum.
 2. Adepth selective method of measuring pH in animal tissue according toclaim 1, wherein said computing comprises computing a numberrepresenting a ratio of concentrations of a protonated and adeprotonated version of a chemical substance that are determined tocontribute to the Raman spectrum and generating pH information on thebasis of said number.
 3. A method according to claim 2, wherein thechemical substance is a form of Urocanic acid.
 4. A method according toclaim 2, wherein the chemical substance is a form of Histidine.
 5. Adepth selective method of measuring pH inside animal tissue according toclaim 2, wherein the number representing the concentrations ofprotonated and deprotonated versions of the chemical substance arecomputed by fitting weights so as to minimize a difference between themeasured spectrum and a simulated spectrum that includes weightedcontributions of a first spectrum and second spectrum of the chemicalsubstance for different pH values.
 6. A method according to claim 5,wherein the first and second spectra are spectra of a form of Urocanicacid.
 7. A method according to claim 1, wherein the tissue is skintissue.
 8. An apparatus for depth selectively measuring pH inside animaltissue, the apparatus comprising a depth selective Raman spectrometer; acomputing device arranged to compute a pH value using a function thatassigns a pH value as a function of a pH dependent aspect of themeasured Raman spectrum; an output for outputting a signal representingthe computed pH value.
 9. An apparatus according to claim 8, whereinsaid computing device is arranged to compute a number representing aratio of concentrations of a protonated and a deprotonated version of achemical substance from the Raman spectrum and generating pH informationon the basis of said number.
 10. An apparatus according to claim 9,wherein the chemical substance is a form of Urocanic acid.
 11. Anapparatus according to claim 9, wherein the number representing theconcentrations of protonated and deprotonated versions of the chemicalsubstance are computed by fitting weights so as to minimize a differencebetween the measured spectrum and a simulated spectrum with weightedcontributions of a first spectrum and second spectrum of the chemicalsubstance for different pH values.
 12. An apparatus according to claim11, wherein the first and second spectra are spectra of a form ofUrocanic acid.
 13. A computer program product comprising instructionsfor computing a pH value using a function that assigns a pH value as afunction of a measured Raman spectrum and outputting pH information onthe basis of said number.
 14. A computer program product according toclaim 13, wherein said computing comprises computing a numberrepresenting a ratio of concentrations of a protonated and adeprotonated version of a chemical substance from the Raman spectrum andgenerating pH information on the basis of said number.
 15. A computerprogram product according to claim 14, wherein the number representingthe concentrations of protonated and deprotonated versions of thechemical substance are computed by fitting weights so as to minimize adifference between the measured spectrum and a simulated spectrum withweighted contributions of a first spectrum and second spectrum of thechemical substance for different pH values.
 16. A method of measuring anindication of a potentially health damaging effect of UV irradiation inskin tissue, the method comprising measuring a Raman spectrum of a partthe tissue selected dependent on a depth from a surface of the tissue;computing an effect of UV irradiation using a function of UV irradiationdependent aspects of the measured Raman spectrum.
 17. A method accordingto claim 16, for measuring formation of cis-UCA in skin tissue as theindication of potentially health damaging effect wherein said computingcomprises computing a number representing a ratio of concentrations of aform of cis-UCA and a reference substance that are determined tocontribute to the Raman spectrum and generating irradiation informationon the basis of said number.
 18. A method according to claim 17, wherethe reference substance is a form of trans-UCA.
 19. An apparatus formeasuring effects of UV irradiation inside skin tissue, the apparatuscomprising a depth selective Raman spectrometer; a computing devicearranged to compute an effect of UV irradiation using a function UVdependent aspects of the measured Raman spectrum; an output foroutputting a signal dependent on the computed effect.
 20. An apparatusaccording to claim 18, wherein said computing device is arranged tocompute a number representing a ratio of concentrations of acontributions of cis-UCA and trans-UCA to the Raman spectrum andgenerating the irradiation information on the basis of said number.